Stat3 activation links a C/EBPδ to myostatin pathway to stimulate loss of muscle mass

Abstract

Catabolic conditions like chronic kidney disease (CKD) cause loss of muscle mass by unclear mechanisms. In muscle biopsies from CKD patients, we found activated Stat3 (p-Stat3) and hypothesized that p-Stat3 initiates muscle wasting. We created mice with muscle-specific knockout (KO) that prevents activation of Stat3. In these mice, losses of body and muscle weights were suppressed in models with CKD or acute diabetes. A small-molecule that inhibits Stat3 activation produced similar responses, suggesting a potential for translation strategies. Using CCAAT/enhancer-binding protein δ (C/EBPδ) KO mice and C2C12 myotubes with knockdown of C/EBPδ or myostatin, we determined that p-Stat3 initiates muscle wasting via C/EBPδ, stimulating myostatin, a negative muscle growth regulator. C/EBPδ KO also improved survival of CKD mice. We verified that p-Stat3, C/EBPδ, and myostatin were increased in muscles of CKD patients. The pathway from p-Stat3 to C/EBPδ to myostatin and muscle wasting could identify therapeutic targets that prevent muscle wasting.

Figure 1. Inflammatory cytokines and p-Stat3 are elevated in muscles of patients with CKD

A. Immunostaining of muscle sections for IL-6 and TNFα(brown color) from biopsies of age- and gender matched, healthy control subjects (left panel) and CKD patients (middle panel). Staining quantification is calculated as the percentage of muscle fibers that are immunostained (right panel; n=3 control subjects; n=4 CKD patients; ruler =50µm). B. Representative western blots for p-Stat3 in control subjects and CKD patients (upper panel) and the ratio of the intensity of p-Stat3 to total Stat3 (lower panel) (n=6 control subjects; n=6 CKD patients). C. Muscle sections from control subjects and CKD patients were immunostained for p-Stat3 (upper panel). Brown nuclei are p-Stat3 positive (arrows). Percentage of p-Stat3 positive nuclei in a total of 550 nuclei (lower panel; n=4 control subjects; n=6 CKD patients). Values are means±SEM. *p<0.05 vs. control subjects. (see also Figure S1, Table S1)

Figure 2. Muscle-specific Stat3 knockout in mice suppresses CKD or streptozotocin-induced muscle wasting

A. Density of p-Stat3 corrected for total Stat3 in lysates of gastrocnemius muscles (upper panel; n=5 mice/group; *p<0.05 vs. sham control mice). Also shown are representative western blots of p-Stat3 (lower panel). B. Changes in body weights of Stat3 KO and Stat3^flox/flox, control mice over 5 weeks following creation of CKD (n=10 pairs of mice; *p<0.05 vs. Stat3^flox/flox). C & D. Average weights of mixed fiber gastrocnemius and tibialis anterior (TA) muscles (n=10 mice/group). E&F. EDL muscles from sham or CKD mice and either Stat3^flox/flox or Stat3 KO were isolated. Rates of protein synthesis (E) and protein degradation (F) were measured (n=20 EDL muscles from 10 mice/group). G. Muscle force of each mouse used in figure 2B was measured on four consecutive days. The average muscle force (in Newtons) is shown (n=10 mice/group). H. Representative western blots of p-Stat3 in lysates of gastrocnemius muscles of acutely diabetic (STZ) and control mice. Bar graph shows the densities of p-Stat3 corrected for that of Stat3 (n=10 mice/group; *p<0.05 vs. CTRL mice). I&J. Average weights of the mixed fiber tibialis anterior (TA) and gastrocnemius muscles from both legs (n=10 mice/group; *p<0.05 vs. control Stat3^flox/flox). Values are means±SEM. (See also Figure S2)

Figure 3. A small molecule inhibitor of Stat3 activation, C188-9, blocks CKD-induced muscle wasting

A. Sham or CKD mice were treated with C188-9 or D5W (diluent) for 14 days. Representative western blots of p-Stat3, Stat3 and GAPDH from lysates of gastrocnemius muscles are shown (n=8 mice/group). B. Differences in body weights of pair-fed, sham or CKD mice treated with C188-9 or D5W at baseline and after 7 or 14 days of treatment (*p<0.05 vs. D5W sham). C&D. Average weights of mixed fiber gastrocnemius and tibialis anterior (TA) muscles from both legs (n=7 mice/group). E. Cryosections of TA muscles were immunostained with anti-laminin to identify the muscle basement membrane. The myofiber areas were measured and the myofiber size distribution was calculated from the areas of ~500 myofibers assessed by an observer blinded to treatment group (n=4 pairs of mice). F. Muscle force of each mouse studied in figure 3C was measured on four consecutive days (Experimental Procedures; n=7 mice/group). G&H. At 2 weeks of C188-9 or D5W treatment, protein synthesis (G) and degradation (H) were measured (n=8 pairs of mice; *p<0.05 vs. D5W). Values are means±SEM.

Figure 4. Stat3 activation in C2C12 myotubes increases the expression of C/EBPδ and myostatin

A. Representative western blots from C2C12 myotubes treated with IL-6 (100 ng/ml) for different times (left panel). Fold-changes in the densities of proteins corrected for GAPDH at different times calculated from values at time zero (right panel), n=3 repeats; *p<0.05 vs. time zero. B. C2C12 myotubes were infected with a lentivirus expressing constitutively active Stat3 (Stat3C-GFP). A representative western blot for the indicated proteins is shown. C. C2C12 myotubes were treated with or without C188-9 for 2 h before adding IL-6 (100 ng/ml) for 24 h. A representative western blot for the indicated proteins is shown. D. C2C12 myoblasts were co-transfected with a plasmid expressing C/EBPδ promoter-driven luciferase, Renila plus a lentivirus expressing Stat3C-GFP and treated with or without IL-6. Dual luciferase activity was measured (n=3 repeats; *p<0.05 vs respective GFP control). E. C2C12 myoblasts were transfected with control siRNA or C/EBPδ siRNA and after differentiation to myotubes were treated with or without IL-6. Representative western blots of Stat3, C/EBPδ and myostatin are shown. F. C2C12 myoblasts were co-transfected with a plasmid expressing the myostatin promoter-driven luciferase plus plasmids (cDNA3 control, Stat3C, C/EBPδ, C/EBPδ siRNA or Stat3C plus C/EBPδ siRNA) and treated with or without IL6. Luciferase activity was measured (n=3 repeats; *p<0.05 vs. cDNA3 CTRL). G. C2C12 myoblasts were transfected with lentivirus expressing a siRNA to myostatin. Myoblasts exhibiting suppression of myostatin were selected and then differentiated after they had been transfected with plasmids expressing Stat3C, C/EBPδ or Stat3C plus C/EBPδ. In these cells, we measured protein degradation (upper panel; n=6 repeats, #p<0.05 vs GFP control, *p<0.05 vs siRNA CTRL). Western blots of proteins expressed in response to tranfections were shown in supplemental figure S7. Values are means±SEM. (See also Figure S3)

Figure 5. Stat3 activation in mouse muscles increases C/EBPδ and myostatin expression

A. Representative western blots of the indicated proteins from lysates of gastrocnemius muscles of control (Stat3^flox/flox) or Stat3 KO sham or CKD mice. B&C. mRNAs of myostatin (B) and C/EBPδ in muscles of sham or CKD mice analyzed by RT-PCR (n=4 mice/group; *p<0.05 vs. Stat3^flox/flox sham). D. Representative western blots of the indicated proteins in lysates of gastrocnemius muscles of STZ vs. WT control mice (n=5 pairs). E. Sham or CKD mice were treated with C188-9 or D5W (diluent) for 14 days. Representative western blots of indicated proteins from lysates of gastrocnemius muscles are shown (n=8 mice/group). F&G. mRNA levels of myostatin (F) and C/EBPδ (G) analyzed by RT-PCR and corrected for GAPDH (n=3 mice/group: wild-type mice without CKD; sham mice treated with C188-9 or D5W; mice with CKD treated with C188-9 or D5W; *p<0.05 vs. WT non-CKD). Values are means±SEM.

Figure 6. C/EBPδ and myostatin mediate CKD or Stat3-induced muscle wasting

A. Body weights of wild type or homo-or hetero-C/EBPδ KO mice following creation of CKD. Values are expressed as a percentage of basal body weight (mean±SEM; n=9 for WT mice; n=11 for C/EBPδ^−/−; n=11 for C/EBPδ^+/− mice; *p<0.05 vs. WT CKD). B. Survival was calculated as the percentage of mice surviving at 3 weeks after CKD or after sham surgery (n=20 for WT; n=25 for C/EBPδ^−/− ; n=21 for C/EBPδ^+/−; *p<0.05 vs. C/EBPδ^{−/ −} CKD). C. Average weights from both legs of red fiber (soleus) or white fiber (EDL) muscles (n=10 mice/group; *p<0.05 vs. WT CKD). D. Representative western blots of p-Stat3 and myostatin from muscles of CKD or sham-operated mice of the following groups: C/EBPδ^{−/ −}, C/EBPδ^+/− or control (WT). E. Cryosections of gastrocnemius muscles from mice that were transfected with lentivirus expressing Stat3C-GPF or GFP and treated with anti-myostatin inhibitor or PBS. The sections were immunostained with p-Smad2/3 (red, lower panel). The upper panel, overlap picture shows GFP-positive myofibers (green) that expressed p-Smad2/3. F. GFP-positive areas in myofibers (Figure 6E) were measured and the mean myofiber sizes of each group are shown. G. The percentage of p-Smad2/3 positive nuclei to total nuclei was calculated. Values are means±SEM. (See also Figure S4).

Figure 7. Evidence for a p-Stat3, C/EBP δ and myostatin pathway in muscles of patients with CKD

A. Representative western blots of p-Akt from muscle biopsies of healthy control or CKD patients. Bar graph shows the densities of p-Akt corrected for GAPDH (lower panel; n=4 CKD patients and 3 healthy subjects). B. Levels of mRNAs of C/EBP δ or myostatin were analyzed by RT-PCR from muscle biopsies of healthy control or CKD patients (n=5 control subjects and 9 CKD patients). C. Representative western blots of the indicated proteins from muscle biopsies from healthy control or CKD patients. D. The band densities were quantified after correction for GAPDH (n=3 pairs for CEBP δ; n=8 pairs for myostatin). Values are means±SEM. *p<0.05 vs. healthy controls.